1. Field
The invention is in the field of fixing biological smears, such as blood smears, to slides for examination and evaluation.
2. State of the Art
There are currently various procedures used to fix biological smears, such as blood smears, on slides and then staining the smears so that certain components of the smears will take on certain characteristics and become visible to a person examining the smear. The fixing of the smear is usually necessary prior to staining so that the stain does not wash the smeared material from the slide.
It is currently normal practice with blood smears to place a smear on a slide and allow the smear to dry. The smear is then fixed on the slide by wetting the smear with anhydrous methanol and allowing the methanol to dry. The smear may be wetted with methanol by dipping, flooding, or spraying the slide. When the methanol evaporates or dries, the smear is fixed on the slide and subsequent staining of the smear does not remove the smear. The fixing of the smear and subsequent staining can be done manually, but there are several commercially available automated staining machines.
When fixing a blood smear on a slide in a humid atmosphere, there is a common problem with water condensing on the smear during fixing of the smear. The presence of water during methanol fixation produces refractile body artifacts (water spots) in the erythrocytes. These water spots persist through staining of the smear and cover items of interest in the smear. Further, they are distracting to the person evaluating the smear. In some cases, the water spots may interfere with diagnosis.
Another problem that occurs when fixing and staining blood smears is that some cellular components of the smear may be poorly preserved by the fixation. Because of this, these components may not show up adequately with subsequent staining of the smear.
A common practice to prevent water spotting of a smear and to stabilize cellular components is to add a stabilizing agent to the fixative liquid to form a fixative solution. A commonly used stabilizing agent, particularly when methanol is used as the fixative liquid, is polyvinyl pyrrolidone (PVP). PVP is commercially available and is a component in the commercially available blood smear fixative sold under the name Diff 3 Fixative System by Coulter Electronics of Hialeah, Fla.
Automated slide staining equipment is currently commercially available. The method of operation of such equipment varies with some equipment mechanically dipping a slide into a reservoir of the solution to be applied to the slide, such as a fixative liquid, otherwise passing the slide through such liquid to be applied, or otherwise mechanically flooding the slide with such liquid. In one commercially available automated slide stainer, sold under the trademark Aerospray by Wescor, Inc., Logan, Utah, slides to be stained are placed in the equipment and stain is sprayed onto the slides. The Aerospray incorporates an automatic fixing cycle so that slides with smears thereon are placed in the machine, a fixative liquid, such as methanol, is first sprayed onto the slides and allowed to evaporate to fix the smear, and then the stain is sprayed onto the slides to accomplish the staining. This equipment also contains a cleaning cycle in which the fixative liquid is used to flush out the staining nozzles. In this equipment, the spray compartment is usually cool and humid and water spotting during fixing can be a serious problem. While the fixative solutions using PVP or other stabilizers work well for hand fixing of smears and in automated equipment using dipping or similar methods for coating a slide, the PVP in its natural state is a solid material and when the methanol evaporates from the methanol-PVP solution, a hard plastic residue is formed. This can cause problems with the use of a methanol-PVP fixative solution, or any other similar fixative solution, in the automated slide staining equipment described wherein the fixative solution is sprayed onto the slides. Methanol is very volatile and evaporates rapidly leaving behind the solid residue. This can clog the nozzles of the equipment and/or distort the spray patterns of the nozzles thereby producing irregular staining or fixing results. It is thus not recommended to use stabilizing agents in fixative liquids used in such automated equipment. Current recommended procedure with such equipment is to fix the smear to the slides by hand so a stabilizing agent can be used without clogging the equipment and then perform the staining using the equipment.
It would be desireable to be able to use the indicated automated staining equipment for the whole process, i.e., the fixing of the smear as well as the staining of the smear, and, at the same time, avoid the water spotting problem during fixing of the smear and enable the stabilization of certain cellular components in the smear while not worrying about clogging the nozzles and passages of the equipment.